The properties of long-range GABAergic (LRG) neurons in the neocortex are poorly defined. Although neocortical LRG neurons have been found in mice, cats, and monkeys, their small numbers make them difficult to study in the intact brain. Optogenetic approaches that specifically label LRG projections can make studies of their connectivity and function experimentally tractable. Indeed, we have recently used optogenetics to characterize a novel class of medial prefrontal (mPFC) LRG neurons that project to the nucleus accumbens (NAcc) and can trigger acute avoidance behaviors. This proposal will incorporate in vivo optical imaging and conditioned behavioral paradigms with patch clamp electrophysiology and optogenetics to further investigate the connectivity and behavioral functions of subcortically-projecting LRG neurons. The findings will add to the long-term study of how neurons and circuits give rise to normal and aberrant behavior. First, the local and distant connectivity of mPFC LRG neurons will be examined. Patch-clamp electrophysiology, intersectional tracing, optogenetics, and pharmacology will be used to determine whether the same population of mPFC-NAcc LRG neurons also forms local and/or distant cortical circuits, such as baslolateral amygdala. Findings from this aim may establish mPFC LRG neurons as a cellular mechanism for synchronizing far-flung cortical circuits. Second, the roles of mPFC-NAcc LRG neurons on conditioned behaviors will be explored. By modifying the intersectional labeling approach to include a genetically encoded calcium indicator, the activity of mPFC- NAcc LRG neurons can be optically imaged in vivo with fiber photometry during conditioned place preference (CPP) and aversion (CPA). LRG neuron activity will be correlated with the context of the conditioned chamber to determine how these neurons encode valence. Using optogenetics to stimulate or inhibit mPFC-NACC LRG projections, I will also determine if they can influence the acquisition or expression of conditioned behaviors. Results from this aim will inform the role of mPFC LRG neurons in circuits involved in conditioned behaviors. Together these aims will advance our understanding of this poorly understood cell type and identify possible roles for LRG neurons in top-down inhibitory processes.